Most peptide and protein drugs are short-lived and have often a short circulatory half-life in vivo. Considering that peptide and protein drugs are not absorbed orally, prolonged maintenance of therapeutically active drugs in the circulation is a desirable feature of obvious clinical importance.
An attractive strategy for improving clinical properties of protein or peptide drugs is a modification of protein or peptide drugs with polymers e.g. polyalkylene-oxides (Roberts et al., Advan Drug Rev. 54, 459-476 (2002)) or polysaccharides like Polysialic acid (Fernandes et al., Biochim Biophys Acta 1341, 26-34 (1997)), dextranes or hydroxylethyl starch. The modification with poly(ethylene glycol) (PEG) has been known for a while. However, modification of proteins with PEG often leads to reduction of the activity of the protein. Therefore alternative systems were developed allowing the releasable coupling of the polymer to the protein or peptide drug using hydrolysable or degradable chemical linkers (U.S. Pat. No. 6,515,100, U.S. Pat. No. 7,122,189, WO 04/089280, WO 06/138572). The protein-polymer conjugate can be regarded as a prodrug and the activity of the protein can be released from the conjugate via a controlled release mechanism. Using this concepts improved pharmacokinetic properties of the drug can be obtained (Zhao et al., Bioconjugate Chem. 17, 341-351 (2006)).
Therefore, WO 04/089280 suggested the use of a hydrolysable PEG-linker. (All documents cited in the specification are incorporated by reference.)
Tsubery et al., (J Biol Chem. 279, 38118-38124 (2004)) demonstrated a hydrolysable PEG-linker for derivatization of proteins based on the Fmoc (9-fluorenylmethyl carbamate) group. A fluorene group is reacted with maleimidopropionic anhydride and N-hydroxysuccinimide, which is further reacted with poly(ethylene glycol) (PEG) and proteins by their amino groups. However, the synthesis of the hydrolysable linker, named MAL-FMS-OSU (9-Hydroxymethyl-2-(amino-3-maleimido-propionate)-7-sulfo fluorene N-hydroxysuccinimidyl carbonate), suffers from low yield and reduced reproducibility. The key problem with the synthesis according to Tsubery et al. is the introduction of the maleimide group by reaction of 9-Hydroxymethyl-2-amino fluorene with maleimido propionic acid anhydride. In this step undesired side reactions like esterification of the OH group in position 9 occurred. Thus, an improved synthesis containing an additional step for protection of the OH group was developed.